Effect of diazoxide postconditioning on cardiac function and mitochondrial cardiolipin in isolated rat heart

AIM: To investigate the effect of diazoxide (D) postconditioning on Cardiac function and mitochondrial cardiolipin in isolated rat heart and to explore the protective effect of ATP sensitive potassium channel on diazo-xide postconditioning myocardium. METHODS: The myocardial ischemia/reperfusion injury model in isolated rat hearts was established by Langendorff apparatus. The isolated rat hearts were randomized into 4 groups (n=8): control group (control), myocardial ischemia/reperfusion injury group (I/R), diazoxide postconditioning group (I/R+D), 5- hydroxy decanoic acid (5-HD) plus diazoxide postconditioning group (I/R+5-HD+D). The hearts in each group were started with 20 min perfusion for equilibration. The hearts in control group perfused for 70 min; The hearts in I/R group was global ischemia for 40 min after ischemia reperfusion at 4 ℃ ST. Thomas cardioplegia, then reperfusion for 30 min; The hearts in I/R+D group were treated with diazoxide (50 μmol/L) in K-H perfusion for 5 min after global ischemia for 40 min, then reperfusion for 25 min; The hearts in I/R+5-HD+D group were treated with 5-HD (100 μmol/L) in K-H perfusion for 5 min before diazoxide postconditioning, then reperfusion for 20 min. The heart rate, coronary outflow volume, heart function, myocardial enzymes and myocardial mitochondrial cardiolipin at the end of perfusion in each group were determined. RESULTS: Compared with control group and I/R+D group, the heart rate, the concentration of heart phospholipid and the coronary outflow volume were reduced, the heart function was significantly impaired the contents of myocardial enzymes were increased in I/R group. However, no significant difference between I/R group and I/R+5-HD+D group was observed. CONCLUSION: The diazoxide postconditioning protects the myocardium by increasing mitochondrial cardiolipin content, reducing the release of myocardial enzymes, improving heart function and reducing myocardial reperfusion injury. The myocardial protective effect of diazoxide is completely blocked by 5- hydroxy decanoic acid.     

Vasodilatation induced by Jumi extraction and its mechanisms

AIM: The objectives of the present study were to examine the effect of Jumi (JM) extraction on relaxation of isolated rat aortic rings, and to elucidate its mechanisms. METHODS: The thoracic aortic rings with and without endothelium of male Sprague-Dawley rats were mounted on a bath system. Vasodilatation of aortic rings preconstricted with 10-6 mol/L of phenylephrine (PE) was measured. RESULTS: JM extraction (0.5-8 g/L) caused a concentration-dependent relaxation in aortic rings. The extent of relaxation was larger in endothelium-intact aortic rings than that in endothelium-denuded aortic rings. Both L-NAME［a nitric oxide synthase (NOS) inhibitor］ and high potassium (20 mmol/L KCl) partly abolished the relaxation action of JM extraction in endothelium-intact aortic rings. Pretreatment with L-NAME also inhibited the relaxation response to JM extraction in endothelium-denuded aortic rings. After incubation with JM extraction, NOS activities enhanced both in endothelium-intact and endothelium-denuded aortic rings. CONCLUSION: JM extraction causes relaxation of aortic rings through endothelium-dependent and independent pathways. The mechanisms might be involved in NOS and endothelium-derived hyperpolarizing factor.     

Role of calcium activated potassium channel in cardioprotection induced by anoxic postconditioning in isolated rat heart

AIM:To investigate whether calcium activated potassium channel (KCa) and mitochondrial permeability transition pore (mPTP) contribute to cardioprotective effect elicited by anoxic postconditioning.METHODS:The isolated perfused hearts of male Sprague-Dawley rats were subjected to 30 min global anoxic followed by 120 min reoxygenation. Formazan content of myocardium was measured at 490 nm spectrophotometrically,and the level of lactate dehydrogenase (LDH) in the coronary effluent was detected. The absorbance of isolated heart mitochondria at 520 nm was determined. RESULTS:Anoxic postconditioning increased formazan content,reduced LDH release,improved the hemodynamic parameters of the left ventricular developed pressure,maximal rise/fall rate of left ventricular pressure,left ventricular end-diastolic pressure and rate pressure product and attenuated the decrease of coronary flow during reperfusion. Pretreatment with paxilline (1 μmol/L) inhibited the effect of anoxic postconditioning. The opening of mPTP was suppressed in the mitochondria isolated from A/R hearts treated with anoxic postconditioning.CONCLUSION:The findings indicate that in the isolated rat heart,anoxic postconditioning protects myocardium against anoxic/reoxygenation injury via inhibiting KCa and the mPTP opening.     

Nitric oxide-mediated the cardioprotection of tumor necrosis factor-alpha on cultured neonatal rat cardiomyocytes during hypoxia/reoxygenation

AIM: To investigate the role of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and protein kinase C (PKC) signaling in tumor necrosis factor-α (TNF-α)-induced cardioprotection against hypoxia/reoxygenation (H/R) injury. METHODS: Neonatal rat ventricular myocytes were pretreated with TNF-α or sodium nitroprusside (SNP) or L-arginine (L-Arg), respectively, for 12 h and then subjected to continuous hypoxia for 12 h, followed by reoxygenation for 6 h. The manganese superoxide dismutase (Mn-SOD) activity of the cells was measured after H/R. Myocyte injury was determined by the release of lactic dehydrogenase (LDH). RESULTS: TNF-α (10⁵ U/L) significantly increased the Mn-SOD activity and decreased release of LDH from ventricular myocytes. The cardioprotection against H/R injury was induced by the pretreatment with SNP (5 μmol/L) or L-Arg (5 mmol/L), which was blocked by ODQ (10 μmol/L), the specific sGC inhibitor, and Chel (5 μmol/L), the specific PKC inhibitor. Pretreatment with L-NAME (100 μmol/L), ODQ, Chel, antoxidant 2-MPG (400 μmol/L) or tyrosine kinase inhibitor genistein (50 μmol/L) attenuated the increased Mn-SOD activity and reduced LDH level induced by TNF-α. CONCLUSION: The results suggest that NO may play a role in TNF-α-induced cardioprotection, which is mediated by sGC and PKC.     

Effects of nitric oxide synthase inhibitors on focal cerebral ischemic injury in rats

AIM:To observe the effects of nitric oxide and different isoforms of nitric oxide synthase inhibitors on the focal cerebral ischemic injury in rats. METHODS:After the rat model of focal cerebral ischemia were established with middle cerebral artery occlusion (MCAO), aminoguanidine(AG)and N^G-nitro-L-arginine(L-NA )were administrated and the cerebral infarct size, NO production,MDA content, nitric oxide synthase(NOS) and SOD activities in the focal ischemic brain tissues were examined. RESULTS:AG could significantly attenuate the focal cerebral ischemic injury, and L-NA had a protective effect when it was administrated at 1 h,6 h but not at 3 h after surgery.CONCLUSION:Cerebral ischemic injury could be attenuated by both selective and nonselective inhibition of NOS.     

Cardioprotection of ischemic postconditioning against ischemia/reperfusion injury in isolated rat hearts

AIM: To investigate the protective role of postconditioning in myocardial ischemia/reperfusion in rats and its mechanisms. METHODS: Cardiac contractility was analyzed by the Langendorff method. Infarct size was determined by dual staining with triphenyltetrazolium chloride and Even's blue dye, and the cardiac arrhythmia was evaluated. postconditioning was conducted by 3 cycles of 30 s ischemia followed by 30 s of reperfusion at the beginning of subsequent persistent reperfusion. RESULTS: Left ventricular systolic pressure (LVSP) and maximal rise rate of ventricular pressure (+dp/dt_max) were higher during reperfusion in postconditioning group compared with control. postconditioning reduced the infarct size in ischemia/reperfusion rat hearts. The cardiac arrhythmia score was decreased in postconditioning group in the first 10 min of reperfusion followed by ischemia compared to control group. postconditioning had similar cardioprotective effect as preconditioning. 5-HD, a selective mitochondrial ATP-sensitive potassium channel (mitoK_ATP) inhibitor, blocked the amelioration of contract function provided by postconditioning. It also abolished the protective effect of postconditioning on cardiac arrhythmia score and infarct size. CONCLUSION: The results show that postconditioning has cardioprotective effect and attenuates reperfusion injury in ischemic heart. The effect might be partly through the activation of mitoK_ATP channel.     

Role of mitochondrial permeability transition pore in TNFα-induced cardioprotection in isolated rat hearts subjected to hypoxia and reoxygenation

AIM: To investigate whether tumor necrosis factor α (TNFα) pretreatment can inhibit mitochondrial permeability transition pore opening in isolated rat hearts subjected to hypoxia and reoxygenation. METHODS: Isolated perfused rat hearts were subjected to 30 min regional hypoxia (occlusion of left anterior descending artery) and 120 min reoxygenation. The infarct size, lactate dehydrogenase (LDH) release during reoxygenation and ventricular hemodynamic parameters were measured. RESULTS: Pretreatment with TNFα at concentration of 1×104 U/L for 7 min followed by 10 min washout reduced the infarct size and LDH release, and improved the left ventricular performance (left ventricular developed pressure and rate-pressure product) and left ventricular end-diastolic pressure during hypoxia and reoxygenation. Administration of atractyloside (Atr, an opener of mitochondrial permeability transition pore, 20 μmol/L) for 20 min (last 5 min of hypoxia and first 15 min of reoxygenation) and paxilline (Pax, a calcium activated potassium channel antagonist, 1 μmol/L) for 5 min before hypoxia attenuated the reduction of infarct size and LDH release and improved the left ventricular performance induced by TNFα. CONCLUSION: The findings indicate that in the isolated rat heart model, TNFα protects myocardium against hypoxia and reoxygenation injury via inhibiting mitochondrial permeability transition pore opening as well as activating calcium, activated potassium channel.     

Proteomic study of myocardial mitochondria with ischemia/reperfusion injury and pinacidil postconditioning in isolated rat hearts

AIM: To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics. METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury. Sprague-Dawley rats were randomly divided into 2 groups: pinacidil postconditioning group (Pina group) and ischemia/reperfusion injury group (I/R group). After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group. In Pina group at the end of 40 min global ischemia, the isolated hearts were perfused with K-H solution containing pinacidil (50 μmol/L) for 2 min followed 58-min perfusion with regular K-H solution. Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots over 2 times were evaluated by a software. Then they were subjected to in-gel digestion, and analyzed by spectrometry. RESULTS: The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH1 were decreased in Pina group compared with I/R group. Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ. The expression levels of one spot was elevated, while the other was decreased. CONCLUSION: Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning.     

Role of heme oxygenase-1 in cardioprotection against anoxia/re oxygenation induced injury and itsmechanisms

AIM:To investigate the role of HO-1 in pro tection of rat hearts against anoxia/reoxygenation-induced injury and its under lying mechanism.METHODS:Cardiac contractility,lactate dehydrogenase (LDH) and infarct area were analyzed by the Langendorff method in isolated rat hearts.RESULTS:After intraperitoneal injection of HO-1 inducer hemin,CO concentration in rat blood enhanced (P<0.01 vs control group).Pretreatm ent with hemin prevented the increase in LVEDP and decrease in LVDP,±dp/d tmax during the anoxia and reoxygenation period in hearts.Hemin had n o effect on changes of coronary flow,but it really inhibited the release of LDH from anoxia/reoxygenation hearts.Hemin also reduced the infarct area in anoxia heart after 2 h reoxygenation (P<0.01).CO concentration in rat blood redu ced after intraperitoneal injection of HO-1 inhibitor ZnPP (P<0.01 vs contr ol group).ZnPP aggravated the decrease in LVDP and ±dp/dtmax.Co mpared with anoxia/reoxygenation heart,pretreatment of ZnPP enhanced the LDH re lease and enlarged the infarct area (P<0.05).GC inhibitor methylene blue a nd cyclooxygenase-2 (COX-2) inhibitor celecoxib both partly abolished the protec tion effect of hemin on LVEDP,LVDP and ±dp/dtmax.Pretreatment o f methylene blue or celecoxib also cancelled the inhibition of LDH release and r eduction of infarct area caused by hemin (P<0.05).CONCLUSION:HO-1 inducer hemin protects heart from anoxia/reoxy genation-induced injury.The cardiac protection of HO/CO might be through GC pathway,and the activation of COX-2 might be also involved in this process.     

Inhibition of calcineurin is involved in cardioprotection induced by ischemic postconditioning in rats

AIM: To demonstrate the mechanisms underlying cardioprotection induced by ischemic postconditioning (I-postC) via studying the alteration of calreticulin (CRT)/calcineurin (CaN) signaling pathway in rat heart subjected to ischemia/reperfusion (I/R).METHODS: The model of myocardial I/R injury in vivo was made by occluding the left anterior descending artery for 45 min followed by 24 h of reperfusion in Wistar rats.Hemodynamics and activity of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in plasma were measured.Myocardial infarct size was measured by 2,3,5- triphenyltetrazolium chloride (TTC) staining and cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL).The activity of CaN,the expressions of CaN and CRT in myocardium were detected by enzyme reaction phosphorus measurement and Western blotting analysis,respectively.RESULTS: Cyclosporin A,the inhibitor of CaN,limited significantly myocardial infarct size and cardiomyocyte apoptosis induced by I/R,but had no significant effect on cardiac function.I-postC ameliorated significantly the cardiac dysfunction induced by I/R.Compared with those in I/R group,the myocardial infarct size,the LDH and CK-MB activities in plasma and the cardiomyocyte apoptotic index were significantly reduced in I-postC group.In addition,I/R-induced upregulation of CaN activity,CaN and CRT expression were relieved by I-postC.No significant difference was found between I-postC and ischemic preconditioning groups.I-postC had stronger protective effect on the reperfused heart compared with cyclosporin A.CONCLUSION: The findings indicate that I-postC protects myocardium against I/R injury,at least in part,via inhibiting the CRT/CaN signaling pathway.     

RISK signaling pathway in myocardial preconditioning and postconditioning

The anti-apoptotic pro-survival kinase signaling cascades, phosphatidylinositol-3-OH kinase (PI3K)-Akt and p42/p44 extra-cellular signal-regulated protein kinases (ERK 1/2), which have been termed the reperfusion injury salvage kinase (RISK) pathway, are involved in cellular survival. In myocardial ischemic preconditioning, pharmacological preconditioning, ischemic postconditioning and pharmacological postconditioning, the activation of these kinase cascades at the time of reperfusion has been demonstrated to confer cardioprotection against reperfusion-induced injury. Targeting the RISK signaling pathway may provide a novel strategy to salvaging viable myocardium and limiting infarct size during myocardial ischemia-reperfusion.     

COX-2 inhibitor protects rat heart against oxidative stress through a pathway independent of cyclooxygenase

AIM: To investigate whether nimesulide ［a selective cyclooxygenase 2 (COX-2) inhibitor］ and piroxicam (an inhibitor of COX-1) protect the rat hearts against oxidative stress induced by hydrogen peroxide,superoxide anion or hydroxyl free radical.METHODS: Cardiac contractility,lactate dehydrogenase (LDH) and malondialdehyde (MDA) were analyzed by the Langendorff method in isolated rat hearts.Production of 6-Keto-PGF1α,a marker of COX activity,was measured in isolated rat hearts.RESULTS: Rat hearts were exposed to hydrogen peroxide (H₂O₂),pyrogallol (which produced superoxide anion) or Vit C+Fe2+ (which produced hydroxyl free radical) for 10 min followed by reperfusion for 30 min.H₂O₂ decreased cardiac contractility and increased LDH release,which was inhibited by nimesulide (3 mg/kg) ［LVDP 72%±10% vs 61%±11%,LDH （5.5±2.5）U/L vs （8.0±2.1）U/L,P<0.05］.Piroxicam (3 mg/kg) increased systolic function (LVDP 73%±10% vs 61%±11%,P<0.05),but exacerbated diastolic function ［LVEDP （29.00±5.61）mmHg vs （23.16±3.57） mmHg,P<0.01］ in H₂O₂ treated rat hearts.Nimesulide also protected rat hearts against superoxide anion and hydroxyl free radical injury.Nimesulide and piroxicam had no effect on the content of 6-Keto-PGF_1α in rat hearts.Mitochondrial ATP sensitive potassium channel (mitoK_ATP) inhibitor 5-HD blocked the improvement of contractility (LVDP and ±dp/dt_max) induced by nimesulide in H₂O₂ treated rat hearts (53%±12% vs 69%±3%,58%±11% vs 72%±7% and 37%±8% vs 51%±4% respectively,P<0.01).CONCLUSION: The results suggests that COX-2 inhibitor nimesulide can protect rat hearts against oxidative injury.The protection is independent of COX activity.Activation of mitoK_ATP may be involved in nimsulide-induced cardioprotection in rat hearts.     

Liver X receptors attenuate myocardial ischemia-reperfusion injury through modulating GLUT-4

AIM：To investigate whether liver X receptors (LXRs) attenuate myocardial ischemia-reperfusion (I/R) injury in isolated rat heart through modulating glucose transporter 4 (GLUT-4). METHODS：Isolated rat hearts were used to establish the model of ischemia-reperfusion injury using Langendorff apparatus. The hearts were divided into 7 groups: LXR agonist T0901317 (0.1, 0.5 and 10 μmol/L) pretreatment groups, ischemic preconditioning group, control group, control+DMSO group, and I/R group. The releases of lactate dehydrogenase (LDH) and creatine kinase (CK), the infarct size, the hemodynamic parameters (left ventricle developed pressure,left ventricle end-diastolic pressure, coronary flow and ±dp/dt max), the relative mRNA level of GLUT-4 and the protein content of GLUT-4 in the myocardial cell membrane were compared between these groups. RESULTS：Besides producing hemodynamic disorders, I/R increased the activities of LDH and CK, and the infarct size in model groups. Treatment with T0901317 significantly suppressed ischemia-reperfusion injury-induced increases in LDH and CK, and reduced the infarct size. T0901317 also significantly ameliorated the parameters of haemodynamics. Treatment with T0901317 significantly increased GLUT-4 expression at mRNA and protein levels in the myocardial cell membrane. CONCLUSION：Liver X receptors may attenuate myocardial ischemia-reperfusion injury in isolated rat heart by modulating the expression of GLUT-4.     

Effects of site-specific antibody of Na+-Ca2+ exchanger on isolated rat heart functions/vascular tone and comparison with ouabain and (±)Bay K8644

AIM：To investigate the effects of site-specific (α-1 repeat: 124HNFTAGDLGPSTIVGSAAFNMF145) antibody of Na+-Ca2+ exchanger (NCX) on the isolated rat heart functions and vascular tone and to compare them with the effects of Na+-K+ pump inhibitor ouabain and L-type Ca2+ channel agonist (±)Bay K8644. METHODS：The heart and thoracic aorta were rapidly isolated from the adult rats weighing 250～300 g. The hemodynamic indexes including left ventricular developed pressure, maximal rate of rise/decline of left ventricular pressure (±dp/dtmax) and coronary resistance were observed by Langendorff isolated heart perfusion system. The rings of thoracic aorta were mounted into the organ baths and the vascular tone was determined. RESULTS：At concentrations of 10, 20 and 40 nmol/L, the site-specific antibody of NCX enhanced the cardiac functions in a dose-dependent manner during the perfusion period with no arrhythmia. At the same time, it did not increase the coronary resistance. The antibody had no effect on the vascular tone of isolated thoracic aorta ring at the same concentrations. Ouabain at concnentration of 0.5 mmol/L and (±)Bay K8644 at concentration of 01 mmol/L had similar effects with the site-specific antibody of NCX at concentration of 40 nmol/L on left ventricular developed pressure and +dp/dtmax of isolated rat hearts during perfusion, but (±)Bay K8644 (01 mmol/L) didn’t have significant effect on -dp/dtmax. Ouabain (0.5 mmol/L) even reduced -dp/dtmax as compared with the control. After administration of ouabain (0.5 mmol/L) for 5 min, most isolated hearts appeared premature pulse more than 5 beats/min. (±)Bay K8644 (0.1 mmol/L) did not induce arrhythmia during all the time of exposure. CONCLUSION: The site-specific (α-1 repeat: 124HNFTAGDLGPSTIVGSAAFNMF145) antibody of NCX enhances both systolic and diastolic functions of isolated rat hearts without arrhythmia. Meanwhile, this antibody doesn’t increase coronary resistance during perfusion and vascular tone in isolated thoracic aorta rings.     

Effect of adenosine infusion before ischemic preconditioning on immature myocardial reperfusion injury in neonatal rabbits

AIM and METHODS:To investigate the cardioprotective effect of adenosine infusion before ischemic preconditioning on immature myocardial reperfusion injury in rabbit heart. Isolated perfused working heart model were performed, all hearts were subjected to 2-hour global hypothermic ischemia and received intermittent cold cardioplegia perfusion.RESULTS:During reperfusion, the recovery of left ventricular systolic pressure, left ventricular end-diastolic pressure, +dp/dt_max, and -dp/dt_max of hearts received adenosine infusion before ischemic preconditioning were significantly improved, myocardial adenosine triphosphate and adenosine diphosphate content and superoxide dismutase activity were higher, the leakage of myocardial creatine kinase and the malondialdehyde content were lower, and myocardial water content was obviously less.CONCLUSION:These results suggest adenosine infusion before ischemic preconditioning enhances cardioprotection of ischemic preconditioning against immature myocardial reperfusion injury in the rabbit heart.     

Production of nitric oxide and change of nitric oxide synthase activity in brains mitochondria of the rats with focal cerebral ischemia/reperfusion

AIM and METHOD:To determine the production of nitric oxide(NO) and change of NO synthase(NOS) activity in mitochondria isolated from the rat brains of the ischemia/reperfusion rat model produced by transient occlusion of middle cerebral artery on the following time points:2 h after occlusion of artery and 30 min,2 h, 4h after reperfusion.RESULTS:After the occlusion of middle cerebral artery,the respiratory control rate(RCR) of mitochondria significantly decreased and slightly increased at 4h after reperfusion.Meantime,the production of NO in mitochondria increased significantly.But with the increase of perfusion, production of NO gradually decreased and reached normal level as in the control group.It also shows that cerebral ischemia increased NOS's activity significantly in the mitochondria and still kept a higher level than the control group although it decreased gradually after reperfusion.But the iNOS's activity did not show obvious change.The change of total NOS's activity depends on the change of cNOS's activity.CONCLUSION: The activation of NO/NOS system in the mitochondria might play an important role in the reperfusion injury during reperfusion of ischemic brain.     

Role of mitochondrial calcium uniporter in hypoxic preconditioning

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in the cardioprotection by hypoxic preconditioning (HPC) and its relationship to mitochondrial permeability transition pore (MPTP). METHODS: Intraventricular balloon technique was employed to measure the left ventricular developed pressure (LVDP), the maximum rise/fall rate of left ventricular pressure (±dp/dtmax), and the left ventricular end-diastolic pressure (LVEDP) in Langendorff isolated rat heart. The hypoxia was achieved by ligation of left anterior coronary artery for 30 min followed by release of ligation for 120 min as reoxygenation. Hypoxic preconditioning was set as two episodes of 5 min global hypoxia and 5 min reoxygenation. RESULTS: Both HPC and treatment with ruthenium red (5 μmol/L) during the first 10 min reoxygenation improved recovery of LVDP, ±dp/dtmax and decreased LVEDP, which was associated with reduced infarct size and lactate dyhydrogenase release. These protective effects were attenuated by treatment with spermine (20 μmol/L) during the first 10 min reoxygenation. Administration of cyclosporin A (0.2 μmol/L) during the last 5 min of hypoxia period and first 15 min of reoxygenation period reduced the injury effect by spermine. CONCLUSION: These results indicate that inhibition of MCU is involved in the cardioprotection of HPC via inhibiting MPTP.     

Erythropoietin inhibits the proliferation of neonatal rat cardiac fibroblasts induced by angiotensin II via PI3-K/Akt signaling pathway

AIM: To investigate the effects of erythropoietin (EPO) on the proliferation of rat cardiac fibroblasts induced by angiotensin Ⅱ(Ang Ⅱ) and to identify the roles of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) signaling pathway and nitric oxide synthase (NOS) in this process. METHODS: Neonatal rat cardiac fibroblasts (CFs) were isolated by collgenase, trypsinase and technique of differential attachment. EPO, Ang Ⅱ, LY294002 (an inhibitor of PI3-K), and L-NAME (an inhibitor of NOS) were added in related group respectively. Growth curves of CFs were established by cell counting and methyl thiazolyl tetrazolium (MTT). The levels of nitric oxide (NO), and the activities of NOS and its isoforms were measured by chemical enzymic method. The expressions of Akt, p-Akt, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were detected by Western blotting. RESULTS: Ang Ⅱ markedly enhanced the proliferation of CFs. The NO level in CFs culture fluid was increased and the proliferation of CFs induced by Ang Ⅱ was suppressed by EPO in a dose dependent manner. After 4 d of administrations, the proliferation ratio of CFs was suppressed 24.4%, 41.5% and 50.5% by EPO at doses of 5×103 U/L, 1×104 U/L and 2×104 U/L respectively. The expressions of phosphated Akt, p-Akt, and eNOS were all up-regulated by EPO. The effect of EPO on NO was blocked by LY294002 and L-NAME, and the suppression of CFs proliferation induced by Ang Ⅱ was diminished similarly. However, LY294002 also down-regulated the expression of eNOS but the L-NAME had no effect on it. CONCLUSION: EPO suppresses the proliferation of neonatal rat CFs induced by Ang Ⅱ in dose dependent manner. The suppressive effects may be due to up-regulating the expression of eNOS and enhancing the production of NO via activating the PI3-K/Akt signaling pathway.     

Role of nitric oxide in ischemia/reperfusion injury and ischemic preconditioning

AIM: To clarify the role of nitric oxide(NO) in ischemic preconditioning(IP) and its effects on apoptosis. METHODS: Seventy-two male Wistar rats were divided into the following six groups:ischemia/reperfusion (IR) group,IP group,IR+L-arg group,IP+L-arg group,IR+L-NAME group and IP+L-NAME group,The following changes were measured:cardiac hemodynamic parameters,infarct size,PMNs counting myocardial MPO activity and TUNEL staining.RESULTS: ①L-arg significantly attenuated ischemia/reperfusion-induced heart injury,reduced PMNs infiltration and cardiomyocyte apoptosis.②L-NAME also significantly reduced infarct size,PMNs infiltration and cardiomyocyte apoptosis compared with IR group,however,L-NAME aggravated ischemia/reperfusions-induced cardiac functional injury.③L-arg or L-NAME did not significantly alter the protective effect of ischemic preconditioning. CONCLUSION: Increased production of endogenous NO before prolonged ischemic period can protect hearts and inhibit apoptosis.L-NAME can inhibit iNOS activity and ONOO^- production in reperfusion period to protect heart.     

Role of cAMP signaling in cardioprotective mechanism of ischemic postconditioning

AIM: To assess the role of the cAMP signaling in cardioprotection by brief intermittent ischemia at the time of onset of reperfusion (i.e. postconditioning). METHODS: The model of rat myocardial ischemia/reperfusion (I/R) was used. The left ventricular functions were assessed by measuring the left ventricular developed pressure (LVDP) and the coronary flow (CF). The injury of myocardium was further confirmed by detecting the releases of lactate dehydrogenase(LDH) and creatine kinase(CK) in coronary effluent. The mRNA expression of caspase-3, bcl-2 and bax in myocardium was determined by real-time PCR. RESULTS: I/R treatment led to the decrease in LVDP and CF, and the increase in the releases of CK and LDH in coronary artery effluent. The mRNA expression of caspase-3 and bax/bcl-2 ratio was up-regulated simultaneously. Postconditioning treatment relieved the injury induced by I/R, which was enhanced by the specific phosphodiesterase 4(PDE4) inhibitor rolipram. On the other hand, the specific adenylyl cyclase inhibitor SQ22536 attenuated those protective effects of postconditioning. CONCLUSION: The cAMP signaling participates in the protective effect of postconditioning on heart from I/R injury, and the effect may be associated with the regulation of apoptosis.